RESUMEN
This paper provides guidance on how to assess the safety of foods derived from genetically modified crops (GM crops); it summarises conclusions and recommendations of Working Group 1 of the ENTRANSFOOD project. The paper provides an approach for adapting the test strategy to the characteristics of the modified crop and the introduced trait, and assessing potential unintended effects from the genetic modification. The proposed approach to safety assessment starts with the comparison of the new GM crop with a traditional counterpart that is generally accepted as safe based on a history of human food use (the concept of substantial equivalence). This case-focused approach ensures that foods derived from GM crops that have passed this extensive test-regime are as safe and nutritious as currently consumed plant-derived foods. The approach is suitable for current and future GM crops with more complex modifications. First, the paper reviews test methods developed for the risk assessment of chemicals, including food additives and pesticides, discussing which of these methods are suitable for the assessment of recombinant proteins and whole foods. Second, the paper presents a systematic approach to combine test methods for the safety assessment of foods derived from a specific GM crop. Third, the paper provides an overview on developments in this area that may prove of use in the safety assessment of GM crops, and recommendations for research priorities. It is concluded that the combination of existing test methods provides a sound test-regime to assess the safety of GM crops. Advances in our understanding of molecular biology, biochemistry, and nutrition may in future allow further improvement of test methods that will over time render the safety assessment of foods even more effective and informative.
Asunto(s)
Seguridad de Productos para el Consumidor , Análisis de los Alimentos , Abastecimiento de Alimentos , Alimentos Modificados Genéticamente/efectos adversos , Plantas Modificadas Genéticamente/efectos adversos , Medición de Riesgo/métodos , Animales , Seguridad de Productos para el Consumidor/normas , Análisis de los Alimentos/métodos , Análisis de los Alimentos/normas , Alimentos Modificados Genéticamente/normas , Ingeniería Genética , Humanos , Cooperación Internacional , Plantas Modificadas Genéticamente/genética , SeguridadRESUMEN
The most important results from the EU-sponsored ENTRANSFOOD Thematic Network project are reviewed, including the design of a detailed step-wise procedure for the risk assessment of foods derived from genetically modified crops based on the latest scientific developments, evaluation of topical risk assessment issues, and the formulation of proposals for improved risk management and public involvement in the risk analysis process.
Asunto(s)
Seguridad de Productos para el Consumidor/legislación & jurisprudencia , Abastecimiento de Alimentos , Alimentos Modificados Genéticamente/efectos adversos , Plantas Modificadas Genéticamente/efectos adversos , Política Pública , Medición de Riesgo , Animales , Seguridad de Productos para el Consumidor/normas , Alimentos Modificados Genéticamente/normas , Ingeniería Genética , Conocimientos, Actitudes y Práctica en Salud , Humanos , Cooperación Internacional , Plantas Modificadas Genéticamente/genéticaRESUMEN
We report a patient with lipid-storage myopathy due to multiple acyl-CoA dehydrogenation deficiency (MADD). Molecular genetic analysis showed that she was compound heterozygous for mutations in the gene for electron transfer flavoprotein:ubiquinone oxidoreductase (ETFQO). Despite a good initial response to treatment, she developed respiratory insufficiency at age 14 years and has required long-term overnight ventilation. Thus, MADD is one of the few conditions that can cause a myopathy with weakness of the respiratory muscles out of proportion to the limb muscles.
Asunto(s)
Acil-CoA Deshidrogenasas/genética , Flavoproteínas Transportadoras de Electrones/genética , Proteínas Hierro-Azufre/genética , Errores Innatos del Metabolismo Lipídico/diagnóstico , Errores Innatos del Metabolismo Lipídico/genética , Metabolismo de los Lípidos , Enfermedades Musculares/genética , Mutación , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Acil-CoA Deshidrogenasas/deficiencia , Adolescente , Edad de Inicio , Secuencia de Bases , Western Blotting , Análisis Mutacional de ADN , ADN Complementario/metabolismo , Complejo I de Transporte de Electrón/deficiencia , Complejo I de Transporte de Electrón/genética , Flavoproteínas Transportadoras de Electrones/metabolismo , Femenino , Fibroblastos/metabolismo , Heterocigoto , Humanos , Modelos Genéticos , Datos de Secuencia Molecular , Enfermedades Musculares/diagnóstico , Fenotipo , Respiración ArtificialRESUMEN
This paper is one of several prepared under the project "Food Safety In Europe: Risk Assessment of Chemicals in Food and Diet" (FOSIE), a European Commission Concerted Action Programme, organised by the International Life Sciences Institute, Europe (ILSI). The aim of the FOSIE project is to review the current state of the science of risk assessment of chemicals in food and diet, by consideration of the four stages of risk assessment, that is, hazard identification, hazard characterisation, exposure assessment and risk characterisation. The contribution of animal-based methods in toxicology to hazard identification of chemicals in food and diet is discussed. The importance of first applying existing technical and chemical knowledge to the design of safety testing programs for food chemicals is emphasised. There is consideration of the presently available and commonly used toxicity testing approaches and methodologies, including acute and repeated dose toxicity, reproductive and developmental toxicity, neurotoxicity, genotoxicity, carcinogenicity, immunotoxicity and food allergy. They are considered from the perspective of whether they are appropriate for assessing food chemicals and whether they are adequate to detect currently known or anticipated hazards from food. Gaps in knowledge and future research needs are identified; research on these could lead to improvements in the methods of hazard identification for food chemicals. The potential impact of some emerging techniques and toxicological issues on hazard identification for food chemicals, such as new measurement techniques, the use of transgenic animals, assessment of hormone balance and the possibilities for conducting studies in which common human diseases have been modelled, is also considered.
Asunto(s)
Exposición a Riesgos Ambientales/efectos adversos , Análisis de los Alimentos , Contaminación de Alimentos/prevención & control , Sustancias Peligrosas/toxicidad , Modelos Animales , Toxicología/métodos , Animales , Alimentos , Contaminación de Alimentos/análisis , Enfermedades Transmitidas por los Alimentos/prevención & control , Humanos , Nivel sin Efectos Adversos Observados , Medición de Riesgo , Gestión de Riesgos , SeguridadRESUMEN
Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most frequently diagnosed mitochondrial beta-oxidation defect, and it is potentially fatal. Eighty percent of patients are homozygous for a common mutation, 985A-->G, and a further 18% have this mutation in only one disease allele. In addition, a large number of rare disease-causing mutations have been identified and characterized. There is no clear genotype-phenotype correlation. High 985A-->G carrier frequencies in populations of European descent and the usual avoidance of recurrent disease episodes by patients diagnosed with MCAD deficiency who comply with a simple dietary treatment suggest that MCAD deficiency is a candidate in prospective screening of newborns. Therefore, several such screening programs employing analysis of acylcarnitines in blood spots by tandem mass spectrometry (MS/MS) are currently used worldwide. No validation of this method by mutation analysis has yet been reported. We investigated for MCAD mutations in newborns from US populations who had been identified by prospective MS/MS-based screening of 930,078 blood spots. An MCAD-deficiency frequency of 1/15,001 was observed. Our mutation analysis shows that the MS/MS-based method is excellent for detection of MCAD deficiency but that the frequency of the 985A-->G mutant allele in newborns with a positive acylcarnitine profile is much lower than that observed in clinically affected patients. Our identification of a new mutation, 199T-->C, which has never been observed in patients with clinically manifested disease but was present in a large proportion of the acylcarnitine-positive samples, may explain this skewed ratio. Overexpression experiments showed that this is a mild folding mutation that exhibits decreased levels of enzyme activity only under stringent conditions. A carrier frequency of 1/500 in the general population makes the 199T-->C mutation one of the three most prevalent mutations in the enzymes of fatty-acid oxidation.
Asunto(s)
Acil-CoA Deshidrogenasas/deficiencia , Acil-CoA Deshidrogenasas/genética , Carnitina/análogos & derivados , Carnitina/sangre , Pruebas Genéticas/métodos , Mutación Missense/genética , Acil-CoA Deshidrogenasa , Acil-CoA Deshidrogenasas/química , Acil-CoA Deshidrogenasas/metabolismo , Alelos , Chaperonina 10/genética , Chaperonina 10/metabolismo , Chaperonina 60/genética , Chaperonina 60/metabolismo , Análisis Mutacional de ADN , Estabilidad de Enzimas , Escherichia coli/genética , Exones/genética , Haplotipos/genética , Heterocigoto , Homocigoto , Humanos , Recién Nacido , Espectrometría de Masas , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida/genética , Polimorfismo de Nucleótido Simple/genética , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reproducibilidad de los Resultados , TemperaturaRESUMEN
Acyl-CoA dehydrogenase (ACAD) defects in isoleucine and valine catabolism have been proposed in clinically diverse patients with an abnormal pattern of metabolites in their urine, but they have not been proved enzymatically or genetically, and it is unknown whether one or two ACADs are involved. We investigated a patient with isolated 2-methylbutyrylglycinuria, suggestive of a defect in isoleucine catabolism. Enzyme assay of the patient's fibroblasts, using 2-methylbutyryl-CoA as substrate, confirmed the defect. Sequence analysis of candidate ACADs revealed heterozygosity for the common short-chain ACAD A625 variant allele and no mutations in ACAD-8 but a 100-bp deletion in short/branched-chain ACAD (SBCAD) cDNA from the patient. Our identification of the SBCAD gene structure (11 exons; >20 kb) enabled analysis of genomic DNA. This showed that the deletion was caused by skipping of exon 10, because of homozygosity for a 1228G-->A mutation in the patient. This mutation was not present in 118 control chromosomes. In vitro transcription/translation experiments and overexpression in COS cells confirmed the disease-causing nature of the mutant SBCAD protein and showed that ACAD-8 is an isobutyryl-CoA dehydrogenase and that both wild-type proteins are imported into mitochondria and form tetramers. In conclusion, we report the first mutation in the SBCAD gene, show that it results in an isolated defect in isoleucine catabolism, and indicate that ACAD-8 is a mitochondrial enzyme that functions in valine catabolism.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/enzimología , Errores Innatos del Metabolismo de los Aminoácidos/genética , Isoleucina/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Oxidorreductasas/deficiencia , Valina/metabolismo , Empalme Alternativo/genética , Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Preescolar , Consanguinidad , Análisis Mutacional de ADN , Estabilidad de Enzimas , Exones , Femenino , Heterocigoto , Humanos , Intrones , Masculino , Mitocondrias/enzimología , Mitocondrias/metabolismo , Mutación/genética , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Pakistán , Transporte de Proteínas , Eliminación de Secuencia/genética , TransfecciónRESUMEN
We developed a PCR detection method that selectively recognizes a single biological control agent and demonstrated that universally primed PCR (UP-PCR) can identify strain-specific markers. Antagonistic strains of Clonostachys rosea (syn. Gliocladium roseum) were screened by UP-PCR, and a strain-specific marker was identified for strain GR5. No significant sequence homology was found between this marker and any other sequences in the databases. Southern blot analysis of the PCR product revealed that the marker represented a single-copy sequence specific for strain GR5. The marker was converted into a sequence-characterized amplified region (SCAR), and a specific PCR primer pair was designed. Eighty-two strains, isolated primarily from Danish soils, and 31 soil samples, originating from different localities, were tested, and this specificity was confirmed. Two strains responded to the SCAR primers under suboptimal PCR conditions, and the amplified sequences from these strains were similar, but not identical, to the GR5 marker. Soil assays in which total DNA was extracted from GR5-infested and noninoculated field soils showed that the SCAR primers could detect GR5 in a pool of mixed DNA and that no other soil microorganisms present contained sequences amplified by the primers. The assay developed will be useful for monitoring biological control agents released into natural field soil.
Asunto(s)
Marcadores Genéticos/genética , Gliocladium/clasificación , Gliocladium/aislamiento & purificación , Control Biológico de Vectores , Reacción en Cadena de la Polimerasa/métodos , Microbiología del Suelo , Cartilla de ADN/genética , ADN Bacteriano/análisis , Gliocladium/genética , Datos de Secuencia Molecular , Especificidad de la EspecieRESUMEN
The question of whether temporal equivalence can be established between test species and humans and be useful in the safety assessment of food additives has puzzled risk assessors throughout decades. The basic biological elements in any mammalian species, including humans, such as homeostasis, basal metabolism and body size/surface area, reproduction features, the timing of cellular proliferation, and aging and health as well as the relation between aging and the diet are essential in this discussion. It is concluded that exposure studies covering selected segments of the total lifetime of any animal species cannot replace lifetime studies in the same animal species in the routine safety testing of food additives, but they may in many cases turn out to become the pivotal study for the entire safety assessment.
Asunto(s)
Aditivos Alimentarios/toxicidad , Pruebas de Toxicidad/métodos , Animales , Muerte Celular/fisiología , División Celular/fisiología , Senescencia Celular/fisiología , Dieta , Modelos Animales de Enfermedad , Aditivos Alimentarios/metabolismo , Homeostasis/fisiología , Humanos , Longevidad , Reproducción/fisiología , Medición de Riesgo/métodos , Especificidad de la Especie , Factores de TiempoRESUMEN
Children are not little adults. Children may respond differently from adults because they are in a state of growth and development; or because of differences in toxicokinetics or toxicodynamics. Infants and children are often assumed to be more susceptible to toxic effects, but this generalization is founded on assumptions rather than on facts. Available data are mostly concerned with toxicity and therapeutic effects of pharmaceuticals, while the effects in children of industrial chemicals are less well documented. Childhood is characterized by growth and development. Toxicants may interfere with these processes, and therefore toxic exposure may have more serious consequences for children than for adults, irrespective of sensitivity. Immature physiological functions of the foetus and young child theoretically make these age groups more vulnerable to toxicants, at least up to 1 year of age. The existing data on effects of chemical exposure in children point in the direction that susceptibility depends on the substance and on the exposure situation. For a particular compound children may be more sensitive than adults, or they may be less sensitive. Further, the sensitivity of children to a particular substance varies greatly with age. It is necessary to view premature neonates, neonates, infants, and children of different ages as separate risk groups. The long-term studies used as the basis for establishing ADIs cover lifetime for laboratory animals. Methods which have special emphasis on reproductive cells, on the foetus, and on the immature organism are used. Taken together, these studies cover exposure during all life stages. However, some specific types of effects, and delayed effects of perinatal exposure are not always included in standard toxicity test protocols. Exposure may also differ between children and adults. The food intake of children is qualitatively and quantitatively different form that of adults, and the EU Scientific Committee for Food has recommended that intake assessment of children be considered separately from that of adults because patterns of consumption are different. The ADI should cover the entire population including children. Special considerations regarding the use of food additives do apply to infants below the age of 12 weeks, who depend entirely on infant formula for nutrition.
Asunto(s)
Aditivos Alimentarios/efectos adversos , Adulto , Factores de Edad , Animales , Biotransformación , Niño , Preescolar , Dieta , Aditivos Alimentarios/metabolismo , Humanos , Lactante , Recién Nacido , Absorción Intestinal , Concentración Máxima Admisible , Neoplasias/inducido químicamente , Conejos , Ratas , Xenobióticos/metabolismoRESUMEN
Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most commonly recognized defect of mitochondrial beta-oxidation. It is potentially fatal, but shows a wide clinical spectrum. The aim of the present study was to investigate whether any correlation exists between MCAD genotype and disease phenotype. We determined the prevalence of the 14 known and seven previously unknown non-G985 mutations in 52 families with MCAD deficiency not caused by homozygosity for the prevalent G985 mutation. This showed that none of the non-G985 mutations are prevalent, and led to the identification of both disease-causing mutations in 14 families in whom both mutations had not previously been reported. We then evaluated the severity of the mutations identified in these 14 families. Using expression of mutant MCAD in Escherichia coli with or without co-overexpression of the molecular chaperonins GroESL we showed that five of the missense mutations affect the folding and/or stability of the protein, and that the residual enzyme activity of some of them could be modulated to a different extent depending on the amounts of available chaperonins. Thus, some of the missense mutations may result in relatively high levels of residual enzyme activity, whereas the mutations leading to premature stop codons will result in no residual enzyme activity. By correlating the observed types of mutations identified to the clinical/biochemical data in the 14 patients in whom we identified both disease-causing mutations, we show that a genotype/phenotype correlation in MCAD deficiency is not straightforward. Different mutations may contribute with different susceptibilities for disease precipitation, when the patient is subjected to metabolic stress, but other genetic and environmental factors may play an equally important role.
Asunto(s)
Acil-CoA Deshidrogenasas/deficiencia , Acil-CoA Deshidrogenasas/genética , Heterocigoto , Mutación , Acil-CoA Deshidrogenasa , Acil-CoA Deshidrogenasas/metabolismo , Adolescente , Alelos , Western Blotting , Chaperonina 10/genética , Chaperonina 10/metabolismo , Chaperonina 60/genética , Chaperonina 60/metabolismo , Niño , Preescolar , Activación Enzimática , Escherichia coli/genética , Exones , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Linaje , Fenotipo , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Análisis de Secuencia de ADN , Eliminación de Secuencia , TemperaturaRESUMEN
The diversity of novel foods and novel ingredients covered by the scope of the EU regulation is such that a check list approach to safety evaluation is inappropriate. Rather, a case-by-case approach is required taking into account the composition of the novel food, its intake, its role in the diet and the intended target group. The SAFEST approach provides a means of targeting the safety evaluation on those aspects, nutritional or toxicological, of a novel food which are of particular concern. Using this approach, novel foods are assigned to one of three classes on the basis of certain background information. For those novel foods which can be shown to be in SAFEST class 1, namely those which are substantially equivalent to a traditional counterpart, no further information is required to demonstrate their safety. For those novel foods in SAFEST class 2, i.e. those sufficiently similar to a traditional counterpart or differing from it only in particular, well defined, characteristics, the evaluation will focus on those differences. Only in the case of novel foods which are not in class 1 or class 2 is extensive testing of the whole food likely to be required. Even in these cases, the testing should follow a scientifically-based hierarchical approach involving: literature reviews; chemical analysis; appropriate in vitro and in vivo tests; and, if necessary, confirmation of safety and nutritional value in humans. Examination of the causes of any adverse effects reported by consumers after the novel food or ingredient has been approved and is introduced into the market may provide additional reassurance of safety.
Asunto(s)
Análisis de los Alimentos , Manipulación de Alimentos/normas , Animales , Contaminación de Alimentos , Hipersensibilidad a los Alimentos , Inspección de Alimentos/normas , Alimentos Formulados/normas , Guías como Asunto , HumanosRESUMEN
Very-long-chain acyl-CoA dehydrogenase (VLCAD) is one of four straight-chain acyl-CoA dehydrogenase (ACD) enzymes, which are all nuclear encoded mitochondrial flavoproteins catalyzing the initial step in fatty acid beta-oxidation. We have used the very fast, Rapid Amplification of cDNA Ends (RACE) based strategy to obtain the sequence of cDNAs encoding human VLCAD from placenta and fibroblasts. Alignment of the predicted amino acid sequence of human VLCAD with those of the other human ACD enzymes revealed extensive sequence homology. Moreover, human VLCAD and human acyl-CoA oxidase showed extensive sequence homology corroborating the notion that these genes are evolutionarily related. Southern blot analysis of genomic DNA from hybrid cell lines was used to localize the VLCAD gene to human chromosome 17p11.2-p11.13105. Using Northern and Western blot analysis to investigate the tissue specific distribution of VLCAD mRNA and protein in several human tissues we showed that VLCAD is most abundant in heart and skeletal muscle. This agrees well with the fact that cardiac and muscle symptoms are characteristic for patients with VLCAD deficiency. Northern blot analysis and sequencing of cloned PCR amplified VLCAD cDNA from four unrelated patients with VLCAD deficiency showed that VLCAD mRNA was undetectable in one patient and that the other three have mutations in both VLCAD alleles. Western blot analysis of patient fibroblasts showed that the identified mutations result in severely reduced amounts of VLCAD protein. None of the patients harbored identical mutations suggesting that the mutational heterogeneity in VLCAD deficiency is large.
Asunto(s)
Acil-CoA Deshidrogenasas/genética , Cromosomas Humanos Par 17 , Mutación , Acil-CoA Deshidrogenasa de Cadena Larga , Acil-CoA Deshidrogenasas/deficiencia , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , Preescolar , Mapeo Cromosómico , Clonación Molecular , Cartilla de ADN , ADN Complementario , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
We identified three overlapping human expressed sequence tags with significant homology to the E. coli ClpP amino sequence by screening the EMBL nucleotide database. With this sequence information we applied 5' and 3'-rapid amplification of cDNA ends (RACE) to amplify and sequence human clpP cDNA in two overlapping fragments. The open reading frame encodes a 277 amino acid long precursor polypeptide. Two ClpP specific motifs surrounding the active site residues are present and extensive homology to ClpP's from other organisms was observed. Northern blotting showed high relative expression levels of clpP mRNA in skeletal muscle, intermediate levels in heart, liver and pancreas, and low levels in brain, placenta, lung and kidney. By analysis of human/rodent cell hybrids the human clpP gene was assigned to chromosome 19.
Asunto(s)
Adenosina Trifosfatasas , Cromosomas Humanos Par 19 , Serina Endopeptidasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , ADN Complementario , Endopeptidasa Clp , Humanos , Células Híbridas , Datos de Secuencia Molecular , Oligonucleótidos Antisentido , Roedores , Homología de Secuencia de Aminoácido , Lugares Marcados de Secuencia , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismoRESUMEN
Novel or new foods may be those introduced for the first time from other parts of the world or they may result from the application of new or modified physical, chemical, and biotechnological treatments performed separately or combined. In addition to offering new possibilities of food for a growing human population, the development raises questions on the criteria for the acceptance of such foods. Conceptual, ethical, and technological issues are involved and call for a world-wide debate on a new and broader GRAS concept, including not only toxicological and nutritional elements but also cultural and philosophical values.
Asunto(s)
Alimentos/efectos adversos , Alimentos/toxicidad , Animales , Alimentos/normas , HumanosRESUMEN
The development of interactive patterns in the first year of life has been studied in healthy mother-infant pairs and in infants whose mothers have psychiatric or psychosocial problems, interfering with their relation to the child. During the first 3 months a relationship develops in healthy mother-infant pairs characterized by an intimate interchange of signals and responses between mother and child. The baby develops a basic pattern of social relating. During the next months the baby begins to expect the mother to fulfil it's wishes and still the mother adapts to and supports the baby's expanding capacities and needs. A beginning sense of self and not-self develops. At 10-12 months the infant is able to move around trying to examine everything within range. At this age the infant senses that there exist other wills and minds than it's own. The child looks at the mother and reads her expressions for "yes", "no", and "maybe". The mother can stop or encourage her child by verbal and body signals. At 12-18 months the child has developed a secure attachment apt to protect the child against later adversities. The development during the first year of life may be seriously disturbed if the mother cannot adapt to and support the baby's signals and needs. Relationship disturbances in the first year of life may be related to maternal or infant problems: Low birth weight babies, irritable babies with low stimulus tolerance and babies with withdrawal reactions following maternal alcoholism or drug abuse during pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Desarrollo Infantil , Bienestar Materno , Relaciones Madre-Hijo , Adulto , Crianza del Niño/psicología , Crianza del Niño/tendencias , Dinamarca , Salud de la Familia , Femenino , Promoción de la Salud , Humanos , Lactante , Recién Nacido , Salud Mental , Desarrollo de la PersonalidadRESUMEN
Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common defect in mitochondrial beta-oxidation in humans. It is an autosomal recessive disorder which usually presents in infancy. The disease manifests itself in periods of metabolic stress to the beta-oxidation system and may be fatal. Four years ago we identified a prevalent disease-causing mutation (G985) which causes an amino acid change (K304E) in the mature MCAD protein. Using a Polymerase Chain Reaction (PCR) based assay for this mutation we have demonstrated: 1. that the G985 mutation is present in 90% of the disease alleles from patients from all over the world; 2. that the allele frequency of G985 in the general population from most European countries is very high (the carrier frequency ranges from 1/68 to 1/333); 3. that MCAD deficiency is not, as has previously been suggested, related to Sudden Infant Death Syndrome (SIDS). Moreover, investigation by Restriction Fragment Length Polymorphism (RFLP) analysis of several families with diagnosed MCAD deficiency revealed that the G985 mutation is only present in chromosomes of a particular RFLP haplotype, suggesting a common chromosomal background for this mutation. The other mutations in the MCAD gene are distributed to all known MCAD RFLP haplotypes. Because 80% of the patients are homozygous for the G985 mutation, DNA based diagnosis of most patients is now fast and easy. In order to make DNA based diagnosis possible for the remaining 20% of patients we have set up PCR/solid-phase based semi-automated sequencing of all 12 exons of the MCAD gene. We have so far identified the mutation in 33 of 45 non-G985 homozygous families with verified MCAD deficiency, thereby bringing the number of known mutations in the MCAD gene up to 26. In order to investigate in detail the molecular defects of the mutant MCAD proteins we overexpressed them in COS-7 and in an E. coli based expression system with and without co-overexpression of the molecular chaperones GroES and GroEL. The expression studies revealed that the primary effect of all the identified mutations is on formation of correct enzyme structure, and does not directly affect the catalytically active regions of the enzyme. We find that our diagnostic set up, consisting of an initial testing by the G985 assay, followed by semi-automated sequencing of DNA from those patients who were indicated to be compound heterozygous, is an important improvement to the diagnosis of MCAD deficiency.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Acil-CoA Deshidrogenasas/deficiencia , Enfermedades Metabólicas/diagnóstico , Acil-CoA Deshidrogenasa , Acil-CoA Deshidrogenasas/genética , Secuencia de Aminoácidos , Humanos , Enfermedades Metabólicas/enzimología , Enfermedades Metabólicas/genética , Mitocondrias/enzimología , Datos de Secuencia MolecularRESUMEN
Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most commonly recognized defect of the mitochondrial beta-oxidation in humans. It is a potentially fatal, autosomal recessive inherited defect. Most patients with MCAD deficiency are homozygous for a single disease-causing mutation (G985), causing a change from lysine to glutamate at position 304 (K304E) in the mature MCAD. Only seven non-G985 mutations, all of which are rare, have been reported. Because the G985 mutation and three of the non-G985 mutations are located in exon 11, it has been suggested that this exon may be a mutational hot spot. Here we describe the results from sequence analysis of exon 11 and part of the flanking introns from 36 compound heterozygous patients with MCAD deficiency. We have identified four previously unknown disease-causing mutations (M301T, S311R, R324X, and E359X) and two silent mutations in exon 11. Our results show that exon 11 is not especially mutation prone. We demonstrate that two of the identified disease-causing mutations can be detected by restriction enzyme digestion of the PCR product from the assay for the G985 mutation, a discovery that makes this assay even more useful than before. On the basis of expression of wild-type and mutant MCAD protein in COS-7 cells, we show that the identified mutations abolish MCAD enzyme activity and that they therefore must be disease causing. The M301T, S311R, and K304E mutations are located in helix H, which makes up part of the dimer-dimer interface of the MCAD tetramer. On the basis of the three-dimensional structure of MCAD and the results from the COS-7 expression experiments, we speculate that the primary effect of the M301T and S311R mutations is on correct folding/tetramer assembly, as it has previously been observed for the K304E mutation.
Asunto(s)
Acil-CoA Deshidrogenasas/deficiencia , Acil-CoA Deshidrogenasas/genética , Exones/genética , Mutación Puntual/genética , Acil-CoA Deshidrogenasa , Acil-CoA Deshidrogenasas/análisis , Animales , Secuencia de Bases , Línea Celular , Análisis Mutacional de ADN , Femenino , Expresión Génica , Humanos , Masculino , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Linaje , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/análisis , TransfecciónAsunto(s)
Acil-CoA Deshidrogenasas/deficiencia , Acil-CoA Deshidrogenasas/genética , Heterocigoto , Mutación/genética , ARN Mensajero/metabolismo , Acil-CoA Deshidrogenasa , Acil-CoA Deshidrogenasas/biosíntesis , Alelos , Southern Blotting , Humanos , Reacción en Cadena de la Polimerasa , Mapeo RestrictivoRESUMEN
Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a serious and potentially fatal inherited defect in the beta-oxidation of fatty acids. Approximately 80% of patients with MCAD deficiency are homozygous for a single disease-causing mutation (G985). The remaining patients (except for a few cases worldwide) are compound heterozygous with G985 in one allele. By sequencing of cloned PCR-amplified MCAD cDNA from a G985 compound heterozygous patient, we identified a C-to-T transition at position 157 as the only change in the entire coding sequence of the non-G985 allele. The presence of the T157 mutation was verified in genomic DNA from the patient and her mother by a PCR-based assay. The mutation changes conserved arginine at position 28 (R28C) of the mature MCAD protein. The effect of the T157 mutation on MCAD protein was investigated by expression of mutant MCAD cDNA in COS-7 cells. On the basis of knowledge about the three-dimensional structure of the MCAD protein, we suggest that the mutation destroys a salt bridge between arginine28 and glutamate86, thereby affecting the formation of enzymatically active protein. Twenty-two additional families with compound heterozygous patients were tested in the PCR-based assay. The T157 mutation was identified in one of these families, which had an MCAD-deficient child who died unexpectedly in infancy. Our results indicate that the mutation is rare. It is, however, noteworthy that a homologous mutation has previously been identified in the short-chain acyl-CoA dehydrogenase (SCAD) gene of a patient with SCAD deficiency, suggesting that the conserved arginine is crucial for formation of active enzyme in the straight-chain acyl-CoA dehydrogenases.